1b Glucose Catabolism: Control of Glycolysis (Slides 54-70)

Under "steady state" conditions, glycolysis operates continuously in most tissues
Reactions 1 (hexokinase), 3 (phosphofructokinase), 10 (pyruvate kinase) have very negative delta-G's and, for that reason, are candidates for the flux control points in glycolysis. [As shown in the figure on slide 56] The other 7 reactions operate at or around delta-G = 0 and are concentration dependent. These 7 reactions readily accommodate changes from flux controlled points.
1. 1. Hexokinase (slides 6-7)
  1. 1st step catalyzed by hexokinase: Glucose + ATP → G6P + ADP + H+
  2. Transfer of high energy phosphoryl group from ATP to glucose
  3. Mg+2 cofactor
    1. Mg+2 complexes with ATP forming Mg+2-ATP complex
      1. ATP is a competitive inhibitor to stop hexokinase
  4. Non-specific - Hexokinase phosphorylates glucose, fructose & mannose, but not galactose
  5. Glucose induces huge conformational change in hexokinase
    1. 2 lobes swing together trapping glucose in active site & excluding water
      1. Places ATP close to #6 –OH of glucose
        Catalysis by proximity
        If water present – it reacts with ATP >>>>
  6. Location: everywhere except liver and kidneys (glucokinase in those places)
2. 10. Pyruvate Kinase (slides 34-5)
  1. [other slide show]
3. 3. Phosphofructokinase (slides 15, 54-70)
  1. Major control point for glycolysis in muscle under most conditions
  2. It is a tetramer with 2 conformations T and R
    1. ATP is both substrate and allosteric inhibitor
      1. Each phosphofructokinase subunit has 2 binding sites for ATP
        ATP substrate site – binds ATP equally in either T or R
        ATP inhibitor site – binds ATP exclusively in T state-->
        F6P binds preferentially R state
      2. ADP, AMP and F2,6P are activators by reversing the inhibitory effect of ATP
  3. When there is High [ATP]
    1. Leads to shift to T state and decrease of phosphofructokinase’s affinity for F6P, which shuts down phosphofructokinase by it not binding to F6P
    2. <-- ATP acts as allosteric inhibitor of phosphofructokinase by binding to T state
      1. Allosteric site has larger km than substrate binding site
  4. Graph of phosphofructokinase activity vs. [F6P] --- Figure on Slide 60
    1. With low ATP or no inhibitors, phosphofructokinase activity near maximum
    2. As [ATP] increases, it shifts the kinetics curve to the right (same VMax, but KMs decrease as you increase ATP)
      1. Becomes more sigmoidal (more cooperativity)
      2. Activators, such as AMP, counter ATP effect by binding to R state of phosphofructokinase (shifting equilibrium to R state)
  5. Direct allosteric control of phosphofructokinase by ATP appears to be all that is needed to control glycolysis flux
    1. When [ATP] is high due to low demand of ATP, phosphofructokinase is inhibited and glycolysis flux is low
    2. When [ATP] is low, glycolysis flux is high to replace ATP
    3. Flux thru glycolysis varies by 100x or more (due to AMP changes)
      1. Results from preferential binding to R state
      2. [ATP] varies < 10% between rest & exercise due to due to buffering activity of (1) Creatine kinase (ATP + creatine ←→ creatine~P + ADP) and (2) Adenylate kinase
        Adenylate kinase catalyzes 2 ADP ←→ ATP + AMP
        K = [ATP][AMP]/[ADP]2 = 0.44
        This rapidly equilibrates [ADP] resulting from ATP hydrolysis in muscle contractions
        In muscle: [ATP] ~ 50X [AMP] & ATP ~ 10X [ADP]
        ∴ 10% ↓[ATP] results in 100% ↑[ADP] as result of adenylate kinase & > 400% ↑[AMP]
        ∴ metabolic signal ↓ in [ATP]:
        Too small to relieve phosphofructokinase inhibition (or increase enzyme activity)
        AMP accounts for activation of phosphofructokinase (overcoming of inhibition) K = 0.5
        But is amplified significantly by adenylate kinase reaction which ↑ [AMP] by amount producing much larger ↑ in phosphofructokinase activity
        2 ATP + 2 creatine ←→ 2 creatine~P + 2 ADP ←→ ATP + AMP
        [ATP]↓ pulls equilibrium to right: [ADP]↑ & [AMP]↑
  6. Substrate Cycling
    1. As noted earlier, allosteric control can not account for 100X change in glycolysis flux --->
    2. GENERAL: 2 different enzymes catalyze forward & reverse reaction and can be independently varied in a thermodynamically favorable manner
      1. F6P + ATP → FBP + ADP (ΔG = -25.9kJ/mol)
        2 opposing reactions cycling substrate to an intermediate and back
        Combined opposing reactions produce much greater pathway flux than possible with allosteric regulation of single enzyme
        Cycling appears to be “energetic price” (ATP) muscle pays to be able to switch from resting to maximum activity
        Can turn on one enzyme while we turn off the other (one for glycolysis with the other in gluconeogenesis
        Controlled by level of F2,6P (controlled by cyclic AMP)
      2. In gluconeogenesis, fructose-1,6-bisphosphatase (FBP)
        FBP + H2O → F6P + Pi (ΔG = -8.6 kJ/mol)
        combined reaction: ATP + H2O ←→ ADP + Pi
    3. SPECIFIC: Thermogenesis = Generate body heat thru substrate cycling in liver and muscle (nonshivering thermogenesis)
      1. stimulated by thyroid hormones (which also stimulate metabolism)
      2. Chronically obese tend to have lower metabolic rates & tend to be more cold sensitive – lower rates of nonshivering thermogensis
      3. Muscle contractions of shivering also produces body heat

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1b Glucose Catabolism: Control of Glycolysis (Slides 54-70)

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